Aldosterone and cortisol synthesis regulation by angiotensin-(1-7) and angiotensin-converting enzyme 2 in the human adrenal cortex

Objective: The branch of the renin--angiotensin system constituting angiotensin-(1–7) [Ang-(1–7)], the Ang II type 2 receptor, the Mas receptors and the Ang-(1–7)-forming enzyme ACE-2, by counteracting the Ang II type 1 receptor (AT1R)-mediated effects, are held to be cardiovascular protective in several conditions. However, whether Ang-(1–7) and ACE-2 are detectable in human adrenocortical tissues and whether they affect aldosterone and cortisol biosynthesis was unknown. Methods: We measured angiotensin peptides with liquid chromatography tandem-mass spectrometry and ACE-2 mRNA with digital droplet (dd)PCR in human aldosterone-producing adenoma (APA) and APA-adjacent tissue obtained from patients with primary aldosteronism. We also investigated the effects of Ang-(1–7) and the ACE-2 activator diminazene aceturate (DIZE) on aldosterone synthase (CYP11B2) and 11β-hydroxylase (CYP11B1) gene expression, in the absence or presence of the AT1R antagonist irbesartan, or of the MasR antagonist A779. Results: APA and APA-adjacent adrenocortical tissues express ACE-2 mRNA and contain detectable amounts of Ang II and Ang-(2–8), but not of Ang I, Ang-(1–5), Ang (3–8) and Ang-(1–7). Under unstimulated and Ang II- stimulated conditions Ang-(1–7) did not blunt CYP11B1 and CYP11B2 mRNA. At supraphysiological concentrations (10−4 mol/l), Ang-(1–7) stimulated both CYP11B1 and CYP11B2 mRNA via the AT1R. The ACE-2 activator DIZE increased by 1.5-fold ACE-2 mRNA but did not blunt Ang II- upregulated CYP11B1 and CYP11B2 expression. Conclusion: These results do not support the hypothesis that the ACE-2/Ang-(1–7)/MasR axis play a protective role by counteracting enhanced aldosterone secretion in humans.


INTRODUCTION
T he adrenocortical zona glomerulosa is the main site of expression of aldosterone synthase (CYP11B2), the key mitochondrial aldosterone-forming enzyme, and, therefore, entails the major source of this mineralocorti-coid hormone [1,2]. More than a dozen factors regulate CYP11B2 expression (reviewed in [3]), among which the octapeptide angiotensin (1)(2)(3)(4)(5)(6)(7)(8), better known as Ang II, is regarded as one of the most important [4]. In primary aldoste-ronism, the most common endocrine form of arterial hypertension, excess aldosterone secretion was contended to be independent from renin and Ang II as the patients with primary aldosteronism show very low levels of Ang II in plasma [5,6]. However, the finding of renin in rat zona glomerulosa and in human aldosterone-producing adenoma (APA) [7,8], alongside the detection of angiotensin receptors in such tumors [9], suggested that locally generated angiotensin peptides can be functionally active in these tissues. Nonetheless, there was no information on the presence of Ang II and related angiotensin peptides in human adrenocortical tissues and in adrenal vein blood from primary aldosteronism patients so far.
Given that activation of ACE-2/Ang-(1-7)/MasR/AT2R pathway can protect from arterial hypertension and hypertension-mediated organ damage by counteracting Ang II/ AT1R-mediated effects [17], we hypothesized that it could also be beneficial by blunting aldosterone production in human primary aldosteronism, the paradigm of aldosterone-and salt-dependent hypertension. This study was, therefore, set out to measure angiotensin peptides in plasma and adrenal tissues of primary aldosteronism patients and determine ACE-2 expression and Ang-(1-7) formation in APA and in APA-adjacent tissues obtained ex vivo at surgery. We also sought for ascertaining if Ang-(1-7) could blunt unstimulated or Ang II-enhanced CYP11B2 and if augmented endogenous Ang-(1-7) generation with diminazene aceturate (DIZE), allegedly an ACE-2-activator, affected aldosterone and cortisol biosynthesis under resting conditions and after Ang II-stimulation.

Patients and tissues
We recruited patients with a conclusive diagnosis of primary aldosteronism because of an APA, as established by the 'five corner criteria' [3]. Their main clinical features at baseline and after adrenalectomy are shown in Supplemental Table 1, http://links.lww.com/HJH/B584. We obtained APA tissue and APA-adjacent tissue from informed consenting patients under sterile conditions in the operating room. After cutting the adrenal gland into halves following the equatorial plane of the APA, small pieces of the tumour and adjacent tissue were immediately snap-frozen in isopentane kept on dry ice and then stored in liquid nitrogen in the institutional human adrenal tissue tumour bank. The collection and the use of the adrenal tissues was approved by the Ethics Committee of the University Hospital of Padova (Protocol # 1925P).
Angiotensin peptides in the adrenal gland and adrenal vein blood Angiotensin peptides were measured in the adrenocortical tissues, in plasma collected during adrenal vein sampling (AVS) from each adrenal vein and from the infrarenal inferior vena cava. These measurements were blindly performed at Attoquant Diagnostics GmbH (Vienna, Austria) with liquid chromatography tandem-mass spectrometry analysis (LC-MS/MS), as described in detail [18]. The lower limits of detection for these assays are shown in Table 1.
RAS equilibrium analysis was performed in human heparin plasma pool containing 50 ng/ml rhACE-2 (recombinant human ACE-2; R&D Systems, Vienna, Austria) in the presence and absence of 100 mg/ml DIZE (Sigma-Aldrich, Milan, Italy) in order to clarify if DIZE is an allosteric activator of ACE-2 as described [19].
To investigate the potential cytotoxicity of DIZE, cells were stimulated for 12 h with 10 -5 and 10 -4 mol/l DIZE, and counted after trypsin treatment using the trypan blue assay.

Gene expression analysis
Total RNA was extracted from the human adrenocortical tissues and cells of interest using RNeasy Mini Kit (Qiagen, Milan, Italy) and following a standardized protocol. The quality of the RNA was checked in a Bioanalyzer Agilent 2100 (Agilent Technologies, Santa Clara, California, USA) using the RNA6000 Nano assay (Agilent Technologies). One microgram total RNA was reverse-transcribed with Iscript (Bio-Rad, Milan, Italy) in a final volume of 20 ml.
We obtained absolute quantification of both ACE-2 and ACE-1 mRNA using Digital Droplet PCR system (ddPCR) (Bio-Rad QX200TM; Bio-Rad Laboratories, Segrate, Italy). Droplets were generated using the QX200 Automated Droplet Generator (Bio-Rad Laboratories) and then transferred to a 96-well PCR plate for amplification in the Bio-Rad C1000 (Bio-Rad Laboratories). After PCR, droplets from each sample were streamed in single file through the QX100 Droplet Reader (Bio-Rad Laboratories). Digital Droplet PCR data were analysed with QuantaSoft analysis software (Bio-Rad Laboratories). Absolute levels of the target gene mRNA were expressed as number of copies/mg of RNA.
The relative expression levels of aldosterone synthase (CYP11B2 gene) and 11b-hydroxylase (CYP11B1 gene) mRNA were measured with a real-time RT-PCR and calculated by the comparative Ct (2 -DDCt ) method using porphobilinogen deaminase (PBGD) as housekeeping gene. Data are presented as mean percentage increase AE SD. Differences observed with real-time RT-PCR were confirmed with digital droplet PCR in a subset of these samples.
Primers for amplification of the genes of interest were designed using ProbeFinder Software (Universal ProbeLibrary, Roche, Monza, Italy, Supplemental Table 2, http:// links.lww.com/HJH/B584). Each experiment was performed in duplicate and repeated at least five times.

Immunoblotting
For these studies, samples were obtained from APA tissues (n ¼ 3) and the adjacent normal adrenal gland (n ¼ 3). Immunoblotting for ACE-2 was performed following a standard protocol. Briefly, tissues were homogenized in a lysis buffer (Thermo Scientific, Milan, Italy) and protein concentration was determined in the soluble supernatant with BCA (Thermo Scientific).
After washing, membranes were incubated for 1 h with an antirabbit secondary antibody (P0448; Agilent-DAKO, Milan, Italy), after which the band intensity was measured in a VersaDoc Imaging System (Bio-Rad, Milan, Italy). Images were analysed with Image Processing and Analysis in Java (Image J-NIH). ACE-2 expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH, #2118; Cell Signaling Technology, Milan, Italy) to adjust for small differences in the amount of loaded protein.

Formation of angiotensin-(1-7) in human adrenocortical tissues
To prove the feasibility of measuring Ang-(1-7) formation in APA and APA-adjacent tissues, we measured Ang-(1-7) formation in a metabolic assay. Briefly, tissue protein extracts were spiked with Ang II, in the presence and absence of MLN-4760 (Merck-Millipore, Vienna, Austria), an established ACE-2 inhibitor. Positive control samples included recombinant human (rh)ACE-2 (1 ng rhACE-2/mg protein) and a sample of proteins from tissues spiked with rhACE-2; both of which were analysed in the presence and absence of MLN-4760. Protein concentration was measured with the Bradford method and was set equal for each sample.

Statistical analysis
Statistical analysis was performed with Prism for Mac OS (vers. 9.0, GraphPad Software La Jolla, California, USA). Nonparametric Mann-Whitney test or one-way ANOVA test were used to compare skewed and normally distributed variables, as appropriate.

RESULTS
Angiotensin peptides, and ACE-1 and ACE-2 gene expressions in adrenal gland Ang II and Ang-(2-8) were detected by LC-MS/MS in both APA and APA-adjacent tissues (Table 1 and Supplemental  Table 3, http://links.lww.com/HJH/B584). All the other angiotensin peptides, including Ang I, Ang-(1-7), Ang-(3-8) and Ang-(1-5), were not detectable, because they were below the LLOD of the assay. Ang II and Ang I levels could be measured also in blood from inferior vena cava and in adrenal vein blood draining from APA and the contralateral side, but no differences between sides, even when data were corrected for the selectivity index to avoid a sampling bias because of different proximities of the catheter's tip to the adrenal cortex, were observed.
Ang-(1-7) and the other angiotensins were undetectable in the blood samples (Table 1). ACE-2 mRNA levels, measured with ddPCR, and ACE-2 protein were lower in APA than in APA-adjacent tissues, whereas the expression levels of ACE-1 were similar (Fig. 1).
We also investigated if DIZE affected CYP11B1 and CYP11B2 gene expressions at concentrations ranging from 10 -7 to 10 -6 mol/l alone or in the presence of 10 -7 mol/l Ang II. The significant stimulatory effect of Ang II alone on CYP11B1 and CYP11B2 mRNA (Fig. 2, panel c) was not blunted by concomitant exposure to nontoxic concentrations of DIZE (Fig. 2, panel c).
We also treated a human heparin plasma pool with rhACE-2 to determine if DIZE could act as an allosteric activator of ACE-2. In a RAS equilibrium analysis, we observed that DIZE did not increase rhACE-2-induced Ang II conversion into Ang-(1-7) (Fig. 3), indicating that DIZE does not act as an allosteric ACE-2 activator under these conditions.

DISCUSSION
ACE-2 has become the target of intense investigative efforts after its identification as the receptor for the SARS-Cov-2 [24,25], the coronavirus that infected over 115 million people and killed over 2.55 million worldwide. The present detection of ACE-2 mRNA in human adrenocortical tissues identifies for the first time the adrenal cortex as a potential target of COVID-19 disease, accounting for occurrence of acute adrenocortical failure contributing to death in a subset of infected patients [26].
This studywas set out before the COVID-19 pandemic to seek for a protective role of the noncanonical branch of the renin-angiotensin system in human primary aldosteronism as ACE-2 has been known for years as a key component of the protective branch of the renin-angiotensin system. Specifically, we investigated if ACE-2, its product Ang-(1-7), and activation of the MasR could modulate expression of the cortisol and aldosterone biosynthetic enzymes (CYP11B1 and CYP11B2), thus affecting the main adrenocortical hormones driving pathological changes in multiple diseases.
We used human adrenocortical tissues, which were obtained ex vivo with utmost care to avoid peptide degradation, to investigate the tissue amounts of Ang II. In keeping with results of functional studies [27][28][29][30], and with the detection of renin [8] and the AT-1R in such tumours [9], we detected Ang II also in some APA and APA-adjacent tissues suggesting that the peptide can play a role in human PA. Interestingly, we did find detectable amounts of both Ang I and Ang II in blood draining from the human adrenal glands of primary aldosteronism patients (Table 1), at similar levels from the adrenal gland harbouring the APA and causing primary aldosteronism, and from the 'nonculprit' adrenal gland. These findings can challenge the view that APAs, or at least a subset of them, are autonomous from Ang II.
ACE-2 can generate Ang-(1-9) and Ang-(1-7) from Ang I and from Ang II, respectively, with Ang II being its preferred substrate [31]. Therefore, the findings of ACE-2 mRNA in APA and APA-adjacent tissue suggested a possible modulatory role of the protective arm of the RAS in human primary aldosteronism. However, at variance with this contention we found low levels of Ang I and Ang II in some adrenocortical tissues and no detectable levels in others (Table 1), which might hinder local Ang-(1-7) production, at least to the levels required to play a protective role in PA. Hence, unless ACE-2 levels were markedly high, enough Ang-(1-7) to counteract the enhanced aldosterone secretion that cause high blood pressure and vascular damage in primary aldosteronism could not be generated locally. Our results indicated that these conditions were not met: even though ACE-2 mRNA and protein were detectable in APA and in APA-adjacent tissue, with lower amounts in the former than in the latter tissue (Fig. 1), the absolute amounts of ACE-2 protein were likely too low to ensure Ang-(1-7) formation.
In a functional metabolic in-vitro assay, we could measure MLN-4760-sensitive Ang-(1-7) generation, an unambiguous index of ACE-2 activity when exogenous rhACE-2 was added to protein extracts from our adrenocortical tissues. Importantly, by this assay, in the absence of human recombinant ACE-2, we detected only a scant ACE-2-independent production of the peptide in protein extracts from APA-adjacent and APA tissues.
Noteworthy, the downregulation of ACE-2 in APA paralleled that of the AT2R [9], and collectively suggested no protective effects of the Ang-(1-7)/AT2R axis in hyperaldosteronism. This contention is further supported by our finding that Ang-(1-7) had no effect on CYP11B1 and CYP11B2 expressions, even at a concentration (10 -8 mol/ l) (Fig. 4, panel a) much higher than that (10 -12 mol/l) found in plasma of hypertensive patients [32].
At high concentrations (10 -5 mol/l and up), Ang-(1-7) stimulated CYP11B2 via the AT1R agonist, as this effect was abrogated by irbesartan (Fig. 4, panel c). This indicates loss of Ang-(1-7) selectivity for MasR and AT2R at high concentrations in human adrenocortical cells, in line with what seen in rabbit aortae and in rats in vivo [33].
We used DIZE, an ACE-2 activator, as a tool to investigate the potential protective effect of enhanced formation of endogenous Ang-(1-7). We discovered that at low concentrations, DIZE markedly raised ACE-2 mRNA (Fig. 2, panel a), but not ACE-2 activity: when added to recombinant human ACE2 in human heparin plasma, DIZE had no effect on Ang-(1-7) formation and on the Ang-(1-7)-to-Ang II ratio (Fig. 3). On the whole, these findings challenged the view that DIZE is an allosteric activator ofACE-2, at least in human adrenocortical tissues in agreement with previous findings [38]. Of note, like Ang-(1-7), DIZE did not blunt CYP11B2 and CYP11B1 expressions (Fig. 2,  panel c).
Collectively, our results indicate that the low tissue levels of ACE-2 are insufficient to provide enough Ang-(1-7) generation to modulate aldosterone and cortisol secretion. Thus, they do not support the contention that the protective branch of the renin-angiotensin system can play a major role in the human adrenal cortex. These conclusions are in line with those of Shefer et al. but differ from the weak blunting effect of 10 -8 mol/l Ang-(1-7) on aldosterone secretion and CYP11B2 gene expression induced by 10 -8 mol/l Ang II in HAC15 cells [39], a subclone of NCI-H295R cells for reasons that remain unclear. However, in the study by Itcho et al. [39], the authors did not use purified or and/or synthetic Ang-(1-7) but conditioned medium obtained after 6 h incubation of HAC15 with Ang II, based on the assumption that cells would have generated Ang-(1-7). Certainly, dissecting the modulatory role of Ang-(1-7) in the human adrenocortical gland requires a careful setup of the experimental conditions, and state-of-the-art techniques, as exploited in our study.
On the whole, while not supporting a protective role of the ACE-2/Ang-(1-7)/MasR/AT2R pathway in the human adrenal cortex and in human primary aldosteronism, our findings can be quite relevant clinically, at a stage when the potential usefulness of strategies aimed at enhancing this protective arm of the renin-angiotensin-aldosterone system are being investigated in multiple cardiovascular diseases.